In recent years, scientists and the general public have become increasingly aware that the use of conventional chemical insecticides may have undesirable environmental consequences. These include groundwater contamination, toxicity to non-target organisms such as birds and fish and potential human health hazards arising from acute or chronic exposure. However, the need for effective insect control has not diminished. This has prompted researchers to develop novel agents for insect control including improved microbial insecticides.
The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (hereinafter B.t.). This bacterial agent is used to control a variety of pests, including leaf-eating caterpillars, beetles and mosquitos. U.S. Pat. No. 4,797,279 issued Jan. 10, 1989 to Karamata et al., discloses hybrid bacterial cells comprising the gene coding for B.t. kurstaki delta-endotoxin and the gene coding for B.t. tenebrionis delta-endotoxin and their preparation. The B.t. hybrids are active against pests susceptible to B.t. kurstaki strains as well as against pests susceptible to B.t. tenebrionis strains. Generally, these hybrids have useful insecticidal properties which are superior to those observed by physical mixtures of the parent strains in terms of level of insecticidal activity, spectrum of activity, or both. The insecticidal compositions comprising such microorganisms may be used to combat insects by applying the hybrids in an insecticidally effective amount to the insects or to their environment.
Another derivation from the bacterium B.t. is disclosed in European Patent Application, Publication No. 0 325 400 A1, issued to Gilroy and Wilcox. This invention relates to a hybrid toxin gene which is toxic to lepidopteran insects. Specifically, the invention comprises a hybrid delta endotoxin gene comprising part of the B.t. var. kurstaki HD-73 toxin gene and part of the toxin gene from B.t. var. kurstaki strain HD-1. The hybrid toxin gene (DNA) encoding a protein having activity against lepidopteran insects is disclosed.
The bacterium B.t. has also been utilized for its insecticidal properties as described in European Patent Application, Publication No. 0 340 948, issued to Wilcox, et al. This invention concerns hybrid pesticidal toxins which are produced by the fusion of an insect gut epithelial cell recognition region of a B.t. gene to diphtheria toxin B chain to prepare a hybrid B.t. toxin which is active against lepidopteran insects. It is suggested that the hybrid B.t. gene may be inserted into a plant or cloned into a baculovirus to produce a toxin which can be recovered. Alternatively, the host containing the hybrid B.t. gene can be used as an insecticide by direct application to the environment of the targeted insect.
In the search for insecticidal compounds, scorpion venom has been identified as a possible source of compounds providing insecticidal properties. Two insect selective toxins isolated from the venom of the scorpion Leirus quinquestriatus quinquestriatus are revealed in Zlotkin et al., Arch Biochem. Biophysics, 240:877 (1985). In a study related to their chemical and pharmacological properties, it has been revealed that one toxin induces fast excitatory contractive paralysis of fly larvae and the other induces slow depressant flaccid paralysis, while both affect sodium conductance in neurons.
Canadian Patent 2,005,658 issued Jun. 19, 1990 to Zlotkin et al., discloses an insecticidally effective protein derived from the scorpion Leirus quinquestriatus hebraeous. In this invention, the venom is lyophilized and separated into fractions. The fraction with the highest toxicity to blowfly larvae and the lowest toxicity to mice is subjected to further purification and the final product is referred to as "LqhP35".
Corresponding with the research and development related to various compositions having insecticidal properties, researcher's have worked to develop methods for producing insecticidal genes and introducing these to the target to be protected or into microbial delivery systems. U.S. Pat. No. 4,879,236 issued Nov. 7, 1989 to Smith and Summers, relates to a method for incorporating a selected gene coupled with a baculovirus promoter into a baculovirus genome to produce a recombinant baculovirus expression vector capable of expression of the selected gene in an insect cell. The method involves cleaning baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. To prepare a recombinant transfer vector, the DNA fragment is inserted into a cloning vehicle and then a selected gene is inserted into this modified cloning vehicle such that it is under the control of the polyhedrin promoter. The recombinant transfer vector is then contacted in insect cells with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome. The baculovirus Autographa californica (AcMNPV) and its associated polyhedrin promotor have been found to be useful in producing a viral expression vector capable of extremely high levels of expression of a selected gene in an insect host cell.
The inventors suggest that the expression vector might be used in a system for controlling insects by selecting a gene which produces a protein which is toxic to a specific insect or to a spectrum of insects and cloning that gene into the AcMNPV expression vector. They suggest that the vector could be applied to the plant or animal to be protected. The recombinant virus could invade the cells of the intestinal wall following ingestion by the insect and begin replication.
The use of DNA technology to incorporate a selected toxin within a baculovirus has been described in several publications. Tomalski et al., Nature, 352:82 (1991); Stewart et al., Nature, 352: 85 (1991); and McCutchen et al. , Biotechnology, 9: 848 (1991) .
A further method for producing insecticidal genes and introducing them to the target to be protected is disclosed in Cutler, Ag. Biotech. News, 7:3 (1990). This article teaches that DENA may be electroporated directly into germinating pollen and that pollen may be put back on the flower to form seeds which then grow into transformed plants. This method has been employed successfully in tobacco plants and may be successful in corn and alfalfa as well. This method may be easier than the electroporation of protoplasts because the ultimate goal is to pollinate the flowers and "let the flowers do the work" rather than to regenerate the plant. The process consists of collecting pollen, germinating it in a germinating medium for 30-60 minutes after which the pollen tube will start to come out of the pollen grain, adding the desired DNA to the liquid suspension containing the pollen, administering an electric shock to open the pores of the pollen, washing the excess DNA away and placing the altered pollen under the stigma of a plant and waiting until seeds are formed. This may be an easy method to move any gene into crop plants.
A prokaryotic delivery system is disclosed in U.S. Pat. No. 4,861,595 issued Aug. 29, 1989 to Barnes and Edwards. This invention concerns the use of treated, substantially intact, microbial cells as a delivery system of protein compounds to animals and humans. The microbial cells initially produce a protein intracellularly via a homologous gene. The protein-producing microbe is treated by chemical or physical means while the cell is substantially intact. Manipulation of the treatment process produces a nonproliferative treated microbial cell without significant loss of the activity of the intracellular compound. Since the cell will not replicate and will have a stable cell wall which may then be broken down in a desired area of the digestive system of the animal or human, it allows the timed or targeted release of the products encapsulatable by the subject invention. After suitable treatment, the protein-producing microbial cell itself is used as the delivery system so that purification of the produced compound is not necessary. Any protein, polypeptide, amino acid or compound, including insecticides, that may be produced by microbial means may be the starting material of the invention.
Researchers have also been able to isolate toxins extracted from the venom of spiders. U.S. Pat. No. 4,925,664 issued to Jackson and Parks on May 15, 1990, discloses methods of treating heart and neurological diseases by applying toxins derived from the spiders Agelenopsis aperta and Hololena curta. The toxins are also effective as specific calcium channel or excitatory amino acid receptor blockers that may be used against insects and related pests.
Another study related to the properties of isolated spider venom toxins has revealed the ability of low molecular weight factors isolated from funnel-web spider venoms to reversibly bind to calcium channels. WO 89/07608 issued Aug. 24, 1989 to Cherksey et al., discloses that these active low molecular weight factors reversibly bind to calcium channels with sufficient specificity and affinity to extinguish calcium conductance in neurons and to permit isolation and purification of calcium channel structures. These venoms were found to be toxic to mammals.
Other applications of spider toxins have been discussed in Jackson and Parks, Ann. Rev. Neurosci., 12:405 (1989). This article teaches that there is great heterogeneity in the toxins of different taxa. It recognizes that experiments have suggested species-specific properties of calcium channels and that spider venoms might provide calcium channel antagonists. The spider venoms discussed are found to affect vertebrates. The article also identifies spider venoms as possible sources of insect-specific toxins for agricultural applications.
Adams et al., in Insect Neurochemistry and Neurophysiology (1986]), Borkovec and Gelman eds., Humana Press, New Jersey, p.397, teaches that multiple peptide toxins which antagonize synaptic transmission in insects have been isolated from the spider Agelenopsis aperta.
U.S. Pat. No. 4,855,405 issued Aug. 8, 1989 to Yoshioka et al., discloses a receptor inhibitor obtained from Joro spider, Nephila clavata, venom and its manufacturing method. Yoshioka et al. demonstrate that their toxins show glutamate receptor inhibitory activity in an insect electrophysiological assay.
U.S. Pat. No. 4,918,107 issued Apr. 17, 1990 to Nakajima et al., relates to a compound which has glutamate receptor inhibitor activity, a process for preparing the same and an insecticidal composition containing the same.
As noted above, previous research with arachnid venoms has identified and partially characterized a number of peptide toxins with insecticidal properties or activity in insect neurophysiology assays. In most cases, however, these toxins have had only moderate insecticidal activity when administered to lepidopteran crop tests such as the tobacco budworm, Heliothis virescens. The relative scarcity of venoms with significant activity in Lepidoptera is underscored by the fact that a recent survey of 64 spider and scorpion venoms identified only four with significant activity in the tobacco hornworm, Manduca sexta, and only two with potent activity in both H. virescens and the beet armyworm, Spodoptera exigua. Quistad et al., J. Econ. Entom., 85:33 (1992). The venoms with high potency in all three lepidopteran species are from the medically important brown recluse spider, Loxosceles reclusa and western black widow spider, Latrodectus hesperus. These spiders are not known to prey preferentially on Lepidoptera. They are, like most spiders, general predators of arthropods, readily consuming most species of prey they encounter, including other spiders. Thus, there is no behavioral or ecological basis for predicting that these spiders produce toxins with unusual potency in lepidopteran pests. Interestingly, the same survey included spiders that are known to be successful predators of Lepidoptera in cropping systems, such as Peucetia viridans. Their venoms, however, are among the least potent in the survey, showing only marginal activity in M. sexta.
Although the two most potent venoms found in the survey described above are from medically important spiders, such spiders do not always have highly insecticidal venoms. Venom from the Sydney funnel web spider, Atrax robustus, which has caused a number of human fatalities in Australia, has been included in a recent study of eighteen spider venoms and found to have only moderate insecticidal activity in the oriental cockroach, Blatta orientalis. Nentwig et al., Zool. Jb. Physiol., 96:279 (1992). Thus, it is clear that predation habits, behavior, and medical significance do not provide a reliable basis for predicting the insecticidal potency of a spider venom. The paralytic or insecticidal effects of spider venoms are unpredictable and may only be determined by experimentation.
The venoms of certain agelenid spiders, Agelenopsis aperta and Hololena curta, have been intensively studied in terms of their chemical compositions and insecticidal properties. A number of peptide toxins have been isolated from these venoms and at least two broad classes have been identified. These toxins, however, are only moderately insecticidal in lepidopteran insects and are less potent in Heliothis than in other lepidopterans. The venom of another agelenid, the European common house spider, Tegenaria attica, has been included in at least two comparisons of the relative insecticidal potencies of spider venoms. Friedel and Nentwig, Toxicon, 27:305 (1989). Nentwig et al., supra (1992). In one survey it was the least effective of six venoms tested, while in the other it was among the six weakest venoms of eighteen tested. Thus, spider venom research to date tends to indicate that agelenid venoms generally and those of Tegenaria spp. in particular are not likely to contain unusually potent insecticidal toxins.
As mentioned previously, a combination of problems associated with conventional chemical insecticides, including pest resistance and injurious effects on non-target organisms, there exists a continuing need for the development of novel means of invertebrate pest control.
Arthropod venoms are a potentially important source of novel insecticidal compounds for use in biological insecticides or as research tools for designing better chemical insecticides. Particularly important for the advancement of this field is the discovery of toxins with high selectivity for insects and potent insecticidal activity in economically significant agricultural pests. The toxins isolated from Tegenaria agrestis, as described herein, are more potent in agricultural pest insects than most other arachnid toxins described to date, appear to be highly selective for insects, and appear to have a mode of action unlike any other arthropod toxins described to date.